Peptides are often favored substrates for the investigation of biomolecule interactions, small molecule inhibition of such interactions, and in the elucidation of biochemical pathways. Peptides can be excellent surrogates for proteins of interest since they represent a segment of the protein but are more easily prepared, modified, and analyzed than full proteins. As such, assays and screens utilizing peptides as substrates or probes have become an integral part of the drug discovery and development process from target identification and validation through hit optimization and lead optimization. In addition, peptide-based diagnostics and therapeutics are increasing with peptide-based assays and screens playing an important role in drug development and clinical applications. The development of assays and screens using peptides as probes that provide high quality and high throughput is a continuing area of interest to the life sciences industry.
There are a number of methods used currently for peptide-based assays. The standard methods are solution-phase-based methods using microtiter plates containing a number of wells generally ranging from 96 to 1536. The test solution and compounds along with a labeled peptide probe are added to each well. The label is generally a fluorogenic or chemiluminescent label which is necessary for detection. After the reaction is complete, the plate is scanned and those reactions which were positive can be distinguished from those that are negative through the fluorescent or chemiluminescent label. Microtiter-plate-based solution phase assays can often be conducted with very high throughput resulting in large data sets that are highly data dense. The primary shortcoming of these types of assays; however, is that the data is generally not of high quality.
Low data quality is generally due to the use of the fluorescent or chemiluminescent detection label. The label itself can oftentimes interfere with the native activity or selectivity of the peptide/protein of interest. This results in a high rate of false positives and negatives. In addition, when adding test compounds that are potential drug candidates, fluorescence and chemiluminescence detection can be compromised either by autofluorescence or by interference from the test compound. It is common for 10% fluorescence interference rates to be reported when screening a library of compounds using assays of this type.
In addition, the information gathered by using fluorescent or chemiluminescent detection does not provide any structural information. Depending on the design of the assay, information such as the degree of change imparted on the peptide probe or the exact location of that change is lost. For example, in a protease screen, a variety of putative peptide substrates may be examined using a fluorogenic label. Cleavage of the peptide at the designed amino acid location results in a positive signal. However, if proteolytic activity were to take place concommitently at other sites within the peptide backbone, that would not be detected by the assay. Alternatively, if two putative sites of proteolysis were available on the peptide, a positive signal would indicate that some proteolysis took place, but would not distinguish at which location. So, while the solution-phase-based assays can often be high throughput, they suffer from producing low-quality data which often requires extensive re-testing by other methods in order to confirm the results or to obtain additional information.
In order to overcome some of the shortcomings of solution-phase assays, solid-phase assay methods have been developed. In solid-phase assays, the peptide probes are immobilized, usually through covalent bonding to a surface, for example, the bottom of a well within a microtiter plate. The peptide may or may not be labeled as in the solution-phase assay. The test solution containing the protein or enzyme, test compound, and/or other reaction components is added. Once the reaction is complete, the solution is removed and the well is washed to remove all other components leaving only the immobilized probe peptide. If the peptide was labeled, the fluorescence or chemiluminescent detection can be used as in the solution-phase assay. The major advantage of the solid-phase method is that auto-fluorescent of fluorescence-interfering materials can be washed away reducing the number of false readouts. This does not; however, overcome limitations due to the presence of the label itself which may interfere with the action of the protein nor does it provide additional structural information missing from the solution-phase assay.
Solid-phase assays also allow for the use of other detection methods. One known method is a radiometric method where a radioactive-isotope-labeled atom is incorporated into the peptide either before or after the desired reaction. Popular radioisotopes include 14C, 32P, 25S, and 152I. Radiometric methods are extremely sensitive and can oftentimes be quite specific. The major disadvantages are the special care and precautions necessary when using radioactive materials and the cost of the isotopes which can preclude use in early screening efforts. In addition, radiometric detection provides no structural information, such as the location of the radioisotope incorporation within the peptide, nor does it provide a reliable measure of degree of radioisotope incorporation.
Another known detection method is a coupled assay which utilizes a second reaction in order to introduce a fluorescent or chemiluminescent label. In a coupled assay, label-free peptides can be used as the probe thereby avoiding any questions as to the effect of the label on protein activity. After the desired reaction is complete, a secondary reaction, generally using a labeled specific antibody, is conducted. The specific antibody only binds to the transformation in question, thereby providing the fluorescent signal. A solid-phase assay using a coupled antibody reaction therefore overcomes the problem of the labeled peptide probe and the interference issues often associated with solution-phase assays. Once again; however, it does not provide information-rich data.
In addition, the coupled assay can require introduction of appropriate surface chemistry in order to immobilize the probes. A large number of different surface chemistries have been introduced for probe immobilization including affinity-based bonding, such as biotin-streptavidin bonding and covalent bonding, as in maleimide, Diels-Alder, click chemistry, etc. Non-specific binding is often a problem with these methods, causing binding of other molecules besides the probe molecule to the surface, thereby compromising signal to noise ratio. For example, if the antibody used in the coupled assay non-specifically binds to the surface, then a false positive signal will be received. Also, the coupled assay is dependent on the availability and selectivity of the antibody. In the absence of a highly selective antibody, the assay will once again result in a high number of false readouts. This lack of specific antibodies is in many areas including epigenetics considered to be the biggest shortcomings within the field.
Despite these drawbacks in information quality and robustness, solid-phase assays have found widespread use within the life sciences industry, primarily due to the high throughput that can be achieved, the epitome of which is the microarray where thousands of probes can be applied to a small surface and interrogated at once. Microarrays, including peptide microarrays, have been demonstrated extensively. Beyond the high throughput, the miniaturized format of microarrays allows minimal use of protein, probe, and reagents thereby reducing the overall cost per probe relative to microtiter-plate formats. The microarray format does not overcome the detection problems; however, of other assay formats.
Other label-free detection methods include optical methods, such as surface-plasmon resonance (SPR). SPR provides no structural information but is a sensitive method by which to observe changes in probes or to detect binding events. Even so, there are some transformations, such as phosphorylation, which SPR cannot reliably detect.
In order to address data quality issues, researchers have turned primarily to mass spectrometry (MS) as a label-free detection method. Mass spectrometry is particularly well-suited for peptide probes since it can not only detect both starting probes and products, but also determine the degree of change on the probe and the location of the change. One such known detection method uses histone methyltransferases (HMETs), where a lysine may be singly, doubly, or triply methylated. Another such detection method uses HMETs having various lysines reside, such as on histone 3 (H3). In this case, methylation can occur on either K4 or K9 and can be readily distinguished by MS as opposed to other methods.
While providing high quality data, MS generally suffers from being only low to medium throughput. This is due primarily to the necessary sample preparation and purification in order to remove impurities and other unwanted materials from the reaction which can adversely affect sensitivity and detection. A favored method is to use a solution-phase assay and coupled liquid chromatography and MS (LC/MS). The chromatographic separation purifies the probe of interest so that it can then be analyzed by MS. This method; however, is very low throughput with each sample requiring a minute or more of time to analyze. Other methods, such as multiplexed (MUX) electrospray with parallel LC systems, have reduced the analysis time to as short as 30 seconds/sample, but still are orders of magnitude behind other assay methods. Another recent system includes a microfluidics-based desalting system coupled with MS detection. This system has been reported to process samples at a rate of one every 5-7 seconds which is significantly higher than other MS-based methods but still falls far short of fluorescence or radiometric-based systems.
In order to try and bridge the gap between high throughput and high quality data, others have tried to combine microarrays with MS detection. The primary difficulty in combining microarrays with MS detection is that methods for immobilization of peptides generally utilize covalent bonding formed by reaction of a reactive group on the peptide and a reactive group on the surface. The covalent bond formation allows the peptide to be immobilized in a specific orientation and through a specific location on the peptide, most commonly either the C or N terminus. Most covalently bound peptides, however, cannot be ionized from the surface once the covalent bond is formed making it incompatible with direct MS detection. Non-covalent immobilization methods have also been used to form microarrays but generally lack specific display orientation which can affect substrate activity. There are, however, methods which can combine a microarray format with MS detection.
A primary example of this would be self-assembled monolayer desorption/ionization MS (SAMDI-MS) where peptides are immobilized through the formation of alkanethiol monolayers on a gold surface. The peptides can then be reacted with the test solution of interest. A chemical matrix is then added to induce ionization and the peptide probes are then analyzed by matrix-assisted laser desorption/ionization (MALDI-MS). SAMDI-MS then combines the high throughput of microarrays with the high quality data of MS detection.
The method, however, suffers from a number of deficiencies. First, results can be highly variable and dependent on the choice and application of the matrix. The optimal matrix can vary depending on the monolayer employed and the nature of the probes. Application of the matrix also needs to be precisely controlled in order to avoid inconsistencies and “patches” across the array resulting in areas of poor signal. In addition, the monolayers themselves may not be robust and are subject to degradation at elevated temperatures and upon exposure to UV light. These issues lead to questions regarding long-term storage and scalability, both critical items for commercialization.
There have recently been described efforts at avoiding some of the issues presented by SAMDI-MS. One method is to utilize fluorous immobilization where a perfluorocarbon modified surface is used to immobilize perfluorocarbon modified probes through fluorous partitioning. Fluorous-based microarrays have been formed using small molecules, peptides, and, most commonly, carbohydrates. Direct MS detection has only been demonstrated with carbohydrate arrays. Fluorous immobilization combines aspects of non-covalent and covalent immobilization. The probes are immobilized non-covalently through fluorous partitioning but in a specific display orientation through a specific end of the molecule; a characteristic usually reserved only for covalent bonding motifs. MS detection can then be conducted either through the use of a nano-structure initiated MS (NIMS) or directly off the surface, in some cases by laser ablation without the need of matrix. These non-matrix methods also have the advantage of being highly robust systems that require a minimum of special storage conditions.
A major limitation of many of these MALDI-MS or SAMDI-MS array methods is the inability to directly enrich the samples in the analytes of interest. Analyte enrichment results in greater sensitivity and higher quality spectra and is often conducted prior to array formation thereby resulting in lower throughput. In this way, one of the major advantages of MALDI or SAMDI based methods over LC/MS and microfluidic based methods can be lost.
There has been only one example of a fluorous peptide microarray and it does not utilize MS as the detection method. Nor would it be capable of doing so since all examples of fluorous microarrays with direct MS detection use either a porous surface, such as silica, or a conductive surface, such as alumina. Fluorous immobilization of peptides is not necessarily as straightforward as immobilization of carbohydrates due to the increased variability for peptides in polarity and charge states which influences fluorous partitioning. The variability in charge states is further increased when using peptides with a number of post-translational modifications. The fluorous peptide array is only capable of using fluorescent detection and suffers from all of the limitations noted earlier for fluorescence-based detection methods.
Accordingly, it would be desirable to have materials, methods, and processes that do not suffer from one or more of the above drawbacks.